Two new approaches to improve the analysis of BALB/c 3T3 cell transformation assay data

Validation activities of the BALB/c 3T3 cell transformation assay (CTA) – a test method used for the assessment of the carcinogenic potential of compounds – have revealed the need for statistical analysis tailored to specific features of BALB/c 3T3 CTA data. Whereas a standard statistical approach for the Syrian hamster embryo (SHE) CTA was considered sufficient, an international expert group was gathered by the European Centre for the Validation of Alternative Methods (ECVAM) to review commonly applied statistical approaches for BALB/c 3T3 CTA. As it was concluded that none of the commonly applied approaches is entirely appropriate, two novel statistical approaches were found to be recommended for the evaluation of BALB/c 3T3 CTA data accounting for possible non-monotone concentration–response relationship and variance heterogeneity: a negative binomial generalised linear model with William's-type downturn-protected trend tests and a normalisation of the data by a specific transformation allowing for application of a general linear model that estimates effects assuming a normal distribution with William's-type protected tests. Both approaches are described in this article and their performance and the quality of the results they generate is demonstrated using exemplary data. Our work confirmed that both approaches are suitable for the statistical analysis of BALB/c 3T3 CTA data and that each of them is superior to commonly used methods. Furthermore, a procedure dichotomising data into negatives and positives is proposed which allows re-testing in cases where inconclusive data are encountered. The scripts of the statistical evaluation programs written in R – a freely available statistical software – are appended including exemplary outputs

Short-Term Treatment with Bisphenol-A Leads to Metabolic Abnormalities in Adult Male Mice

Bisphenol-A (BPA) is one of the most widespread endocrine disrupting chemicals (EDC) used as the base compound in the manufacture of polycarbonate plastics. Although evidence points to consider exposure to BPA as a risk factor for insulin resistance, its actions on whole body metabolism and on insulin-sensitive tissues are still unclear. The aim of the present work was to study the effects of low doses of BPA in insulin-sensitive peripheral tissues and whole body metabolism in adult mice. Adult mice were treated with subcutaneous injection of 100 µg/kg BPA or vehicle for 8 days. Whole body energy homeostasis was assessed with in vivo indirect calorimetry. Insulin signaling assays were conducted by western blot analysis. Mice treated with BPA were insulin resistant and had increased glucose-stimulated insulin release. BPA-treated mice had decreased food intake, lower body temperature and locomotor activity compared to control. In skeletal muscle, insulin-stimulated tyrosine phosphorylation of the insulin receptor β subunit was impaired in BPA-treated mice. This impairment was associated with a reduced insulin-stimulated Akt phosphorylation in the Thr308 residue. Both skeletal muscle and liver displayed an upregulation of IRS-1 protein by BPA. The mitogen-activated protein kinase (MAPK) signaling pathway was also impaired in the skeletal muscle from BPA-treated mice. In the liver, BPA effects were of lesser intensity with decreased insulin-stimulated tyrosine phosphorylation of the insulin receptor β subunit